OBJECTIVE: To deliver and overexpress the hsp70 gene in cultured chondrocytes to investigate its effect on nitric oxide (NO)-induced apoptosis of chondrocytes. METHODS: Primary chondrocyte cultures were established from rabbit joints. The cells were transduced with an empty adenovirus vector (Ax1w) or an adenovirus vector harboring the hsp70 E-tag fusion gene (AxSHEwt). Apoptosis was induced by sodium nitroprusside (SNP) dihydrate, which generates NO, or by staurosporine, which is a proapoptotic agent dependent upon Bax or Bak protein. Cell viability and apoptosis induction were estimated by lactate dehydrogenase (LDH) assay, Hoechst 33342 staining, or the TUNEL method. To study Hsp70, cytochrome c, and caspase 3, Western blot analyses were performed. RESULTS: The AxSHEwt-transduced cells escaped apoptosis, as revealed by the LDH assay, Hoechst 33342 staining, and the TUNEL method. A massive amount of the tagged Hsp70 was demonstrated in the AxSHEwt-transduced chondrocytes but not in control cells. Hsp70 did not affect the cytosolic cytochrome c level, but appeared to have obstructed the activation of caspase 3. CONCLUSION: Experimentally overexpressed Hsp70 almost completely inhibited NO- or staurosporine-induced apoptosis in primary chondrocytes.
OBJECTIVE: To deliver and overexpress the hsp70 gene in cultured chondrocytes to investigate its effect on nitric oxide (NO)-induced apoptosis of chondrocytes. METHODS: Primary chondrocyte cultures were established from rabbit joints. The cells were transduced with an empty adenovirus vector (Ax1w) or an adenovirus vector harboring the hsp70 E-tag fusion gene (AxSHEwt). Apoptosis was induced by sodium nitroprusside (SNP) dihydrate, which generates NO, or by staurosporine, which is a proapoptotic agent dependent upon Bax or Bak protein. Cell viability and apoptosis induction were estimated by lactate dehydrogenase (LDH) assay, Hoechst 33342 staining, or the TUNEL method. To study Hsp70, cytochrome c, and caspase 3, Western blot analyses were performed. RESULTS: The AxSHEwt-transduced cells escaped apoptosis, as revealed by the LDH assay, Hoechst 33342 staining, and the TUNEL method. A massive amount of the tagged Hsp70 was demonstrated in the AxSHEwt-transduced chondrocytes but not in control cells. Hsp70 did not affect the cytosolic cytochrome c level, but appeared to have obstructed the activation of caspase 3. CONCLUSION: Experimentally overexpressed Hsp70 almost completely inhibited NO- or staurosporine-induced apoptosis in primary chondrocytes.